Nucleic acid sequence analysis is becoming increasingly important in many research, medical, and industrial fields (see, e.g., Caskey (1987) Science 236: 1223-1228; Landegren et al. (1988) Science, 242: 229-237; Arnheim et al. (1992) Ann. Rev. Biochem., 61: 131-156, etc.). In particular, more than 2,000 conditions have been identified as single-gene defects for which the risk of producing affected offspring can be mathematically predicted. Among these conditions in man include Huntington's chorea, cystic fibrosis, α1 antitrypsin deficiency, muscular dystrophy, Hunter's syndrome, Lesch-Nyhan syndrome, Down's syndrome, Tay-Sachs disease, hemophilias, phenylketonuria, thalasemias, and sickle-cell anemia. In addition to various genetic diseases can be diagnosed utilizing nucleic acid sequence analysis, various infectious diseases can be diagnosed by the presence in a clinical sample of a specific DNA sequence characteristic of the causative microorganism. These include, but are not limited to bacteria, viruses, and parasites. In addition, particular pathogen strains (e.g., drug resistant pathogens) can be identified by nucleic acid analysis. Also the identification of various nucleic acid polymorphisms has utility for basic research, genotyping, and forensics.
Current diagnostic techniques for the detection of known nucleotide differences include: hybridization with allele-specific oligonucleotides (ASO) (Ikuta, et al., Nucleic Acids Research 15: 797-811 (1987); Nickerson, et al., PNAS (USA) 87: 8923-8927 (1990); Saiki, et al., PNAS (USA) 86: 6230-6234 (1989); Verlaan-de Vries, et al., Gene 50: 313-320 (1980); Wallace, et al., Nucleic Acids Research 9:879-894 (1981); Zhang, Nucleic Acids Research 19: 3929-3933 (1991)); allele-specific PCR (Gibbs, et al., Nucleic Acids Research 17: 2437-2448 (1989); Newton, et al., Nucleic Acids Research 17: 2503-2516 (1989)); solid-phase minisequencing (Syvanen, et al., American Journal of Human Genetics 1993; 52: 46-59 (1993)); oligonucleotide ligation assay (OLA) (Grossman, et al., Nucleic Acids Research 22: 4527-4534 (1994); Landegren, et al., Science 241: 1077-1080 (1988)); and allele-specific ligase chain reaction (LCR) (Abravaya, et al. (1995) Nucleic Acids Res. 23: 675-682; Barany, et al. (1991) Proc. Natl. Acad. Sci., USA, 88: 189-193; Wu, et al., (1989) Genomics 4: 560-569). Genomic DNA is analyzed with these methods by the amplification of a specific DNA segment followed by detection analysis to determine which allele is present.
The routine use of nucleic acid amplification reactions for allelic detection/discrimination, particularly in clinical settings, has been hampered because the quantification of nucleic acids is made more difficult or less accurate or both because data captured during amplification reactions are often significantly obscured by signals that are not generated in response to the target nucleic acid (i.e., noise). Furthermore, the data captured by many monitoring methods can be subject to variations and lack of reproducibility due to conditions that can change during a reaction or change between different instances of a reaction.